The focus of our research is towards understanding the mechanisms that underlie ATP-dependent protein remodeling carried out by molecular chaperone machines and the role of chaperones in ATP-dependent proteolysis. Molecular chaperones function during non-stress conditions to facilitate folding of newly synthesized proteins, to remodel protein complexes, and to target regulatory proteins and misfolded proteins for degradation. During cell stress, chaperones play an essential role in preventing folding intermediates from becoming irreversibly damaged and forming protein aggregates. They promote recovery from stress by disaggregating and reactivating proteins, a process once thought to be impossible. They are also involved in delivering damaged proteins to compartmentalized proteases. Protein aggregation, misfolding and premature degradation are major contributors to a large number of human diseases, including cancer, Alzheimers, Parkinsons, type II diabetes, cystic fibrosis, and prion diseases. The goal of our research is to understand how chaperones function and how they act with proteases to provide the foundation for discovering preventions and treatments for these diseases. One goal of our research is to elucidate the mechanism of action of Hsp90. The Hsp90 family of heat shock proteins represents one of the most abundantly expressed and highly conserved families of molecular chaperones. Eukaryotic Hsp90 is known to control the stability and the activity of more than 200 client proteins, including receptors, protein kinases and transcription factors. Hsp90 is also important for the growth and survival of cancer cells and drugs targeting Hsp90 are currently in clinical trials. To gain insight into the mechanism of action of Hsp90 chaperones, we are studying Hsp90 from Escherichia coli, Hsp90Ec. We found that Hsp90Ec promotes reactivation of heat-inactivated proteins in a reaction that requires the DnaK chaperone system. An Hsp90 ATPase inhibitor, geldanamycin, inhibits protein reactivation, as do mutants with substitutions in the Hsp90Ec ATP binding site. These results are the first demonstration of an ATP-dependent chaperone activity for Hsp90Ec. By using mutants in DnaK, we showed that the chaperone activity of DnaK, both ATP hydrolysis and peptide binding, were essential for protein remodeling in collaboration with Hsp90Ec. We were able to divide the reactivation reaction into two steps and demonstrated that the DnaK system acts first on the client protein, and then Hsp90Ec and the DnaK system collaborate synergistically to complete remodeling of the client protein. Further results using a two-hybrid system indicate that Hsp90Ec and DnaK interact in vivo and studies using ultrafiltration provide additional evidence to suggest that the two chaperones also interact in vitro. A second goal is to understand the mechanism of protein remodeling and disaggregation by Clp/Hsp100 molecular chaperones, including ClpB of prokaryotes and its yeast homolog, Hsp104. ClpB/Hsp104 dissolves insoluble aggregated proteins in combination with a second molecular chaperone system, DnaK in E. coli and Hsp70 in yeast. In the absence of the DnaK/Hsp70 system, ClpB and Hsp104 have the intrinsic ability to disaggregate soluble aggregates in vitro. Hsp104 and ClpB share 43% sequence identity and are structural homologs. Both consist of four domains: an N-terminal domain, two nucleotide-binding domains and a middle-domain. Despite the similarities, ClpB and Hsp104 show species specificity for their DnaK/Hsp70 partner. Hsp104 cannot function in E. coli and ClpB cannot act in Saccharomyces cerevisiae. To study the species specificity of ClpB/Hsp104 and DnaK/Hsp70, we exchanged one or more of the four domains of ClpB and Hsp104 by genetic engineering and tested the chimeras both in vivo and in vitro. We found that chimeras with the middle-domain of Hsp104 promote thermotolerance in S. cerevisiae and chimeras containing the middle-domain of E. coli support survival at high temperature in E. coli. In protein reactivation assays in vitro, chimeras containing the Hsp104 middle-domain collaborate with Hsp70 and those with the ClpB middle-domain function with DnaK. Additional experiments showed that the region responsible for the specificity is within helix 2 and helix 3 of the middle-domain. Furthermore, several ClpB mutants containing amino acid substitutions in helix-2 of the middle-domain are defective in protein disaggregation in collaboration with DnaK. In a bacterial two-hybrid assay, DnaK interacts with ClpB and with chimeras that have the ClpB middle-domain, implying that species specificity is due to an interaction between DnaK and the middle-domain of ClpB. Our results suggest that the interaction between Hsp70/DnaK and helix 2 of the middle-domain of Hsp104/ClpB determines the specificity required for protein disaggregation both in vivo and in vitro, as well as for cellular thermotolerance. A third goal is to investigate the mechanism of action of Clp chaperones in proteolysis. Clp proteases of prokaryotes have analogous structures, functions and mechanisms of action to the eukaryotic proteasome. They are composed of an ATP-dependent protein unfolding component and a protease component. We are currently studying ClpX, which associates with a proteolytic component, ClpP, forming the ClpXP ATP-dependent protease. We discovered that ClpXP participates in cell division of E. coli. We found that in vitro E. coli ClpXP degrades FtsZ, the major cytoskeletal protein in bacteria and a tubulin homolog. FtsZ is an essential cell division component, forming a dynamic ring where constriction occurs to divide the cell by polymerizing into fibers and bundles in a GTP-dependent manner. To investigate the role of ClpXP in cell division, we examined the effects of clpX and clpP deletions in several strains that are defective for cell division. Taken together these studies imply that ClpXP may degrade multiple cell division proteins, thereby modulating the precise balance of the components required for division.